Excluding cancers of the skin, breast cancer is the most common cancer among women, accounting for one out of every three cancer diagnoses in the United States. In 1997, approximately 180,200 new cases of invasive breast cancer are expected to be diagnosed, and 43,900 women are expected to die from this disease. Only lung cancer causes more cancer deaths in women.
Currently, the primary method of detecting breast cancer in women is through mammography, or by physical examination. Unlike numerous other cancers, at present no method is available to reliably detect the existence of breast cancer by examining the level of specific blood markers. For example, in the case of prostate cancer, the antigen PSA (for prostate specific antigen) can be detected in the blood and is indicative of the presence of prostate cancer. Thus, the blood of men at risk for prostate cancer can be quickly, easily, and safely screened for elevated PSA levels. No such method currently exists for women at risk of breast cancer. This invention addresses this and other needs.
The present invention provides methods of detecting markers from a biological sample from a patient, wherein the level of the marker indicates the presence of breast cancer in the patient.
In numerous embodiments of the invention, a method for assessing the presence of a breast cancer in a patient will include several steps, including providing a biological sample from the patient, detecting the level of one or more markers in the sample, and comparing the level of the one or more markers with a control level that is representative of a level in a normal, cancer-free patient. Using such methods, an elevation of marker level in a patient compared to the control level indicates the presence of breast cancer in the patient.
In preferred embodiments, the marker used in this invention will be M2 Pyruvate Kinase, or a derivative or fragment thereof. Also preferred is the use of hnRNPK, or derivatives or fragments thereof. The invention also provides methods for identifying novel molecules useful in the assays described herein.
In preferred embodiments, the level of a marker will be measured in a blood sample. The xe2x80x9clevelxe2x80x9d as used herein can refer to MRNA level, DNA level, protein level, enzyme activity, the presence of particular isoforms, or any other marker of gene number, expression, or activity. In particularly preferred embodiments, the protein level of one or more marker described herein will be measured.
In preferred embodiments, the level of marker will be quantitated and compared with a control value or sample. In particularly preferred embodiments, the difference between an elevated level and a control level will be statistically significant.
In numerous embodiments, the present methods will further include an additional step, wherein an additional diagnostic step specific to breast cancer will be performed. For example, following a detection of an elevated level of a marker, which elevated level indicates the presence of a breast cancer, the indication will be confirmed using one or more techniques specific to breast cancer detection, such as mammography, physical examination, biopsy, etc.
Definitions
A xe2x80x9cblood samplexe2x80x9d refers to an amount of blood removed to allow diagnostic analysis of components within the blood. These components may be blood cells, such as lymphocytes or other white blood cells, or may be blood fractions that are partially or completely devoid of cells, e.g., plasma or serum). A blood sample can also refer to cells removed from bone marrow.
When a cell is said to have an xe2x80x9celevated levelxe2x80x9d of a marker, it means that it has a level of the marker that is measurably or detectably higher than the level of the marker in a normal, non-cancerous cell. The difference between the higher level of the marker and the normal level may be based on quantitative or qualitative methods of detection.
The phrase xe2x80x9cdetecting a breast cancerxe2x80x9d refers to the ascertainment of the presence or absence of breast cancer in an animal. xe2x80x9cDetecting a breast cancerxe2x80x9d can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal. Detecting a breast cancer can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the animal.
A xe2x80x9cbreast cancerxe2x80x9d in an animal refers to the presence of cells originating in the breast that possess characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, breast cancer cells will be in the form of a tumor, but such cells may also exist alone within a patient.
xe2x80x9cProviding a biological samplexe2x80x9d means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from a patient, but can also be accomplished by using previously isolated cells (e.g., isolated by another person), or by performing the methods of the invention in vivo.
A xe2x80x9cbiological samplexe2x80x9d refers to a cell or population of cells or a quantity of tissue or fluid from a human. Most often, the sample has been removed from a human, but the term xe2x80x9cbiological samplexe2x80x9d can also refer to cells or tissue analyzed in vivo, i.e. without removal from a human. Often, a xe2x80x9cbiological samplexe2x80x9d will contain cells from the human, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure marker levels.
xe2x80x9cTissue biopsyxe2x80x9d refers to the removal of a biological sample for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.
xe2x80x9cDetecting a level of a markerxe2x80x9d refers to determining the expression level of a gene or genes encoding a target polypeptide. The copy number of a gene can be measured in multiple ways known to those of skill in the art, including, but not limited to, Comparative Genomic Hybridization (CGH) and quantitative DNA amplification (e.g., quantitative PCR). Gene expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of (e.g., gDNA, cDNA, MRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of a target, in particular in comparison with a control level.
To xe2x80x9ccomparexe2x80x9d levels of markers means to detect marker levels in two samples and to determine whether the levels are equal or if one or the other is greater. A comparison can be done between quantified levels, allowing statistical comparison between the two values, or in the absence of quantification, for example using qualitative methods of detection such as visual assessment by a human.
A xe2x80x9ccontrol samplexe2x80x9d refers to a sample of biological material representative of healthy, cancer-free humans. The level of a target in a control sample is desirably typical of the general population of normal, cancer-free humans. This sample can be removed from a patient expressly for use in the methods described in this invention, or can be any biological material representative of normal, cancer-free humans, including cancer-free biological material taken from a human with cancer elsewhere in its body. A control sample can also refer to an established level of a target, representative of the cancer-free population, that has been previously established based on measurements from normal, cancer-free humans.
An xe2x80x9cincreased level of a targetxe2x80x9d means a level of a target polypeptide (e.g. M2PK), that, in comparison with a control level of the target polypeptide, is detectably higher. The method of comparison can be statistical, using quantified values for the level of the target, or can be compared using non-statistical means, such as by visual assessment by a human.
When a level of a target (e.g., M2PK) mRNA, protein, enzyme activity, or copy number is xe2x80x9cmeasured,xe2x80x9d it is assessed using qualitative or quantitative methods. Preferably, the level is determined using quantitative means, allowing the statistical comparison of values obtained from biological samples and control values. The level can also be determined using qualitative methods, such as the visual analysis and comparison by a human of multiple visibly labeled samples, e.g., fluorescently labeled samples detected using a fluorescent microscope or other optical detector (e.g., image analysis system, etc.).
The present invention provides methods for detecting a breast cancer based on detecting the level of any of a number of markers, wherein the level of the marker reflects the presence of breast cancer cells in a patient. This invention is based upon the surprising discovery that certain proteins that have not been previously associated with breast cancer are in fact elevated in tissues of patients with breast cancer, and thus provide methods for diagnosis of breast cancer.
It has previously been identified that the ZNF2 17 gene is elevated in breast cancer cells (see, e.g., WO 98/02537, the gene is referred to as ZABC1 in this document). It has now been discovered that a number of proteins bind to the ZNF217 gene product, and which are themselves elevated in breast cancer cells. These ZNF217-binding proteins are thus markers for breast cancer cells, and can be used for the diagnosis of breast cancer in a patient, alone or in combination with other diagnostic methods.
In particular, two proteins, M2 Pyruvate Kinase (M2PK), and heterogeneous nuclear ribonucleoprotein K (hnRNPK) were found to bind to ZNF217 protein and to be highly expressed in breast cancer cells. The present invention provides methods for diagnosing breast cancer through the detection of the level of the genes encoding these proteins, or through the detection of the level of gene product itself. This invention also provides methods for identifying additional proteins that can be used in the diagnosis of breast cancer.
I. Markers
Any of a number of markers can be used to detect breast cancer according to the present methods. In preferred embodiments, the level of M2 Pyruvate Kinase (M2PK), or any derivative, variation, or fragment thereof, or of Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) can be used.
M2PK (see, e.g., GenBank accession numbers P14786, S30038, A33983) is a glycolytic enzyme that can exist as a tetramer with high affinity for its substrate, phosphoenolpyruvate, or as dimer with low substrate affinity (Eigenbrodt et al., 1997). In tumors the low activity dimeric isoenzyme predominates. The metabolic consequences of this M2PK inactivation include increased aerobic glycolysis, increased biosynthetic capability and decreased requirement for oxygen. Stabilization of M2PK in the inactive form has been associated with HPV16 E7 protein binding to it (Zwerschke et al., 1999). Binding of M2PK by ZNF217 suggests that ZNF217 may mimic E7 and induce a metabolic state permissive for cell proliferation in breast cancer cells.
hnRNPK (see, e.g., Q07244 or NPxe2x80x94002131) is a transcription factor that can modulate expression of genes such as CMYC and thymidine kinase (TK) (Michelotti et al., 1996; Hsieh et al., 1998). The observation that ZNF217 binds hnRNPK indicates that these proteins may collaborate to regulate expression of important growth and immortalization related genes.
In numerous embodiments, the level of M2PK, hnRNPK, or other proteins will be assessed. For example, the level of M2PK mRNA, gene copy number, protein level, or enzyme activity will be assessed using standard techniques. As explained below, preferred means of detecting, e.g., increased M2PK levels is the analysis of protein levels in blood using immunoassays such as ELISA assays, as described below (see, e.g., Eigenbrodt et al. (1997) Anticancer Res. 17:3153-5156.
In addition, any other ZNF217 binding protein, such as splicing factor Srp30c, cDNAs with homology to RNA binding proteins, and laminin binding protein (LBP) can be used. Additional ZNF217-associating molecules can easily be identified using standard techniques, e.g., 2-hybrid screens, GST pull-down, co-immunoprecipitations, affinity chromatography, etc. Preferably, the level of any molecules, e.g., genes or proteins, identified using such methods will be assayed in breast cancer cells, wherein a molecule found to associate with ZNF217 and to be expressed at high levels in breast cancer cells can be effectively used in the present methods.
In addition, the ZNF217 protein can be used (see, e.g., WO 98/02539, wherein the gene is referred to as ZABC1) to detect the presence of a breast cancer. In preferred embodiments, the level of ZNF217 is detected in the blood using an immunoassay, e.g., ELISA.
In numerous embodiments, the level of more than one marker will be detected in a single biological sample. Such combinations of marker detection can be used, e.g., to confirm or refine the diagnostic indication provided by a single marker level alone. For example, the M2PK level, which can reflect the presence of any of several types of cancer, e.g., colon cancer, breast cancer, etc., can be detected simultaneously to or prior to the detection of the ZNF217 level, wherein an elevated level of M2PK and ZNF217 indicates the presence of one type of cancer alone, e.g., breast cancer.
The manipulation of any of the marker genes or proteins described herein, e.g., for the purpose of producing protein or nucleic acids, or for creating variants, derivatives, fragments, etc., of any of the markers, can be accomplished using standard molecular biological techniques, as described, e.g., in Ausubel et al. (ed.) (1990) Current Protocols in Molecular biology, Greene Publishing and Wiley-Interscience, New York, Glover (ed.) (1987) DNA Cloning: A Practical Approach, vols 1-3, IRL Press, Oxford, or Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., vols 1-3, Cold Spring Harbor Press, New York.
II. Treating breast cancer
The markers described herein can also be used to reduce the growth and/or proliferation of breast cancer cells. Such inhibition can be an effective treatment, alone or in combination with other treatments, for breast cancer. For example, ZNF217 and/or M2PK, each of which is present at abnormally high levels in breast cancer cells, can be inhibited using any of a large number of standard techniques, such as antisense, ribozymes, dominant negatives, small molecule inhibitors, antibodies, and others. The selection, isolation, synthesis, and use of such inhibitory techniques is well known to those of skill in the art.
III. Assays of marker levels
As indicated above, assays of the copy number or level of activity or expression of any of the proteins described herein provide a measure of the presence or likelihood of a breast cancer. The sequence of these proteins are known and hence, copy number can be directly measured according to a number of different methods as described below.
A. Detection of Copy Number
In one embodiment, the presence of, or predilection to cancer, is evaluated simply by a determination of the copy number of a marker gene. Methods of evaluating the copy number of a particular gene are well known to those of skill in the art. For example, the genomic location of the M2PK gene, 15q22, is amplified in certain cancer cells and can be detected using the methods provided herein.
1. Hybridization-based Assays
One method for evaluating the copy number of marker-encoding nucleic acid in a sample involves a Southern transfer. In a Southern Blot, the genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.
An alternative means for determining the copy number of any of the marker genes described herein is in situ hybridization. In situ hybridization assays are well known (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the conditions for use vary depending on the particular application.
Preferred hybridization-based assays include, but are not limited to, traditional xe2x80x9cdirect probexe2x80x9d methods such as Southern blots or in situ hybridization (e.g., FISH), and xe2x80x9ccomparative probexe2x80x9d methods such as comparative genomic hybridization (CGH). The methods can be used in a wide variety of formats including, but not limited to substrate(e.g. membrane or glass) bound methods or array-based approaches as described below.
In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.
The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bp to about 1000 bases.
In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block non- specific hybridization.
In comparative genomic hybridization methods a first collection of (sample) nucleic acids (e.g. from a possible tumor) is labeled with a first label, while a second collection of (control) nucleic acids (e.g. from a healthy cell/tissue) is labeled with a second label. The ratio of hybridization of the nucleic acids is determined by the ratio of the two (first and second) labels binding to each fiber in the array. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the marker gene copy copy number.
Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In one particularly preferred embodiment, the hybridization protocol of Pinkel et al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.
2. Amplification-based assays.
In still another embodiment, amplification-based assays can be used to measure copy number. In such amplification-based assays, the marker nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction (PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate (e.g., healthy tissue) controls provides a measure of the copy number of the marker gene.
Methods of xe2x80x9cquantitativexe2x80x9d amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). The known nucleic acid sequence for the markers (see, for MKGenBank Accession Numbers U60669 S78775 and X59506) is sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.
Other suitable amplification methods include, but are not limited to ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990) Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
B. Detection of Gene Expression
As indicated above, the level of any of the present markers can also be assayed as a marker for a predilection to breast cancer.
In preferred embodiments, marker activity is characterized by a measure of marker gene transcript (e.g., MRNA), by a measure of the quantity of translated protein, or by a measure of enzymatic activity (e.g., pyruvate kinase activity in the case of M2-PK, or transcription based assays in the case of hnRNPK).
1. Detection of Gene Transcript.
a) Direct Hybridization Based Assays.
Methods of detecting and/or quantifying the level of a marker gene transcript (marker MRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., vols 1-3, Cold Spring Harbor Press, New York.
For example, one method for evaluating the presence, absence, or quantity of marker cDNA involves a Southern transfer as described above. Briefly, the MRNA is isolated (e.g., using an acid guanidinium-phenol-chloroform extraction method, Sambrook et al. supra.) and reverse transcribed to produce cDNA. The cDNA is then optionally digested and run on a gels in buffer and transferred to membranes. Hybridization is then carried out using the nucleic acid probes specific for the target cDNA.
The probes can be full length or less than the full length of the nucleic acid sequence encoding the protein. Shorter probes are empirically tested for specificity. Preferably nucleic acid probes are 20 bases or longer in length. (See Sambrook et al. for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization.) Visualization of the hybridized portions allows the qualitative determination of the presence or absence of cDNA.
Similarly, a Northern transfer may be used for the detection of an mRNA directly. In brief, the mRNA is isolated from a given cell sample using, for example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. As with the Southern blots, labeled probes are used to identify and/or quantify the mRNA.
b) Amplification-based Assays.
In another preferred embodiment, a marker transcript (e.g., M2-PK mRNA) can be measured using amplification (e.g PCR) based methods as described above for directly assessing copy number of the gene. In a preferred embodiment, a transcript level is assessed by using reverse transcription PCR (RT-PCR). As indicated above, PCR assay methods are well known to those of skill in the art. Similarly, RT-PCR methods are also well known.
C. Detection of Expressed Protein
The xe2x80x9cactivityxe2x80x9d of a marker can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
In a typical embodiment, a marker polypeptide is detected using an immunoassay, such as an ELISA assay. As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte (i.e., the polypeptide). The immunoassay is thus characterized by detection of specific binding of a marker polypeptide to an anti-marker antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.
The polypeptide is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites and Terr (1991) Basic and Clinical Immunology 7th Edition. 
Immunological binding assays (or immunoassays) typically utilize a xe2x80x9ccapture agentxe2x80x9d to specifically bind to and often immobilize the analyte (in this case marker polypeptide or subsequence). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds a marker polypeptide. The antibody (anti-marker) may be produced by any of a number of means well known to those of skill in the art.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled marker polypeptide or a labeled anti-marker antibody. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/marker polypeptide complex.
In one preferred embodiment, the labeling agent is a second human marker antibody bearing a label. Alternatively, the second marker antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, e.g., as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542).
As indicated above, immunoassays for the detection and/or quantification of marker polypeptide can take a wide variety of formats well known to those of skill in the art.
Preferred immunoassays for detecting a marker polypeptide are either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured. In one preferred xe2x80x9csandwichxe2x80x9d assay, for example, the capture agent (anti-marker antibodies) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the marker polypeptide present in the test sample. The marker thus immobilized is then bound by a labeling agent, such as a second human marker antibody bearing a label.
In competitive assays, the amount of analyte (marker polypeptide) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (marker polypeptide) displaced (or competed away) from a capture agent (anti marker antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, marker polypeptide is added to the sample and the sample is then contacted with a capture agent. The amount of marker polypeptide bound to the antibody is inversely proportional to the concentration of marker polypeptide present in the sample.
In one particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of marker polypeptide bound to the antibody may be determined either by measuring the amount of marker polypeptide present in an marker polypeptide /antibody complex, or alternatively by measuring the amount of remaining uncomplexed marker polypeptide. The amount of marker polypeptide may be detected by providing a labeled marker polypeptide.
The assays of this invention are scored (as positive or negative or quantity of marker polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. The intensity of the band or spot can provide a quantitative measure of marker.
Antibodies for use in the various immunoassays described herein, can be produced according to standard methods (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY.
Kits for providing the subject immunoassays are also provided. Generally, such kits comprise at least antibody specific for M2PK, hiRNPK, or any other protein described herein as well as any reagents necessary for the detection of antibody-marker binding complexes. The kits may further comprise other components of the immunoassay, such as solid support, solutions and the like.
It is particularly convenient in a clinical setting to perform the immunoassay in a self-contained apparatus, and such devices are provided by the subject invention. A number of such methods are known in the art. The apparatus will generally employ a continuous flow-path of a suitable filter or membrane, having at least three regions, a fluid transport region, a sample region, and a measuring region. The sample region is prevented from fluid transfer contact with the other portions of the flow path prior to receiving the sample. After the sample region receives the sample, it is brought into fluid transfer relationship with the other regions, and the fluid transfer region contacted with fluid to permit a reagent solution to pass through the sample region and into the measuring region. The measuring region may have bound to it the first antibody, and second labeled antibody combined with the assayed sample and the sandwich assay, e.g., ELISA assay, performed as above.
Such methods can also be used to distinguish between various forms of proteins. For example, M2PK can exist in a tetrameric and a dimeric form, which can be distinguished in a variety of ways, e.g., separation based on size. In such methods, cells can be extracted in a lysis buffer containing, e.g., 100 mM N2HPO4,/NaH2PO4, 1 mM DTT, 1 mM NaF, 1 mM mercaptoethanole, 1 mM xcex5-aminocaproic acid, 0.2 mM PMSF, and 10% glycerol, pH 7.4.
1. Detection of Enzyme Activity.
In another embodiment, marker level (activity) is assayed by measuring the enzymatic activity of the marker polypeptide. Methods of assaying the activity of this enzyme are well known to those of skill in the art. For example, methods of measuring pyruvate kinase activity are well known and described, e.g., in Eigenbrodt et al. (1997), Brinck et al. (1994) Virchows Archiv 424:177-185, and Zweschke et al. (1999) PNAS 96:1291. Typically, M2-PK activity can be measured using high (2 mM) and low (0.2 mM) phosphoenolpyruvate concentrations. Also, glycolytic and glytaminolytic flux measurements can be made (see, e.g., Mazurek et al., (1997).
In addition, methods of detecting hnRNPK activity are well known. For example, hnRNPK cis-activates genes containing a CT promoter element such as CMYC. Thus, e.g., transcription based assays including CT promoters operably linked to reporter genes, e.g., luciferase or GFP, or DNA binding assays using CT promoter elements, can be used. Also, hnRNPK binds to a number of proteins, such as TBP, VAV oncoprotein, and the protein-tyrosine kinases SRC, FYN, and LYN (see, e.g. Ostareck-Lederer et al. (1998)). Protein binding assays using any of these proteins can be performed using standard techniques.
IV. Secondary Screening Steps
In numerous embodiments of the present invention, a secondary screening step will be performed. For example, if a level of one or more of the markers described herein is found to be elevated compared to a control level, then an additional method of detecting breast cancer will be performed to confirm the presence of the breast cancer. Any of a variety of secondary steps can be used, such as mammography, ultrasound, PET scanning, MRI, or any other imaging techniques, biopsy, clinical breast examination, ductogram, nipple discharge examination, or any other method.
V. Breast Cancers
The methods described herein can be used to detect any type of breast cancer. For example, adenocarcinoma, ductal carcinoma in situ (DCIS), infiltrating (or invasive) ductal carcinoma (IDC), infiltrating (or invasive) lobular carcinoma (ILC), inflammatory breast cancer, in situ, lobular carcinoma in situ (LCIS), medullary carcinoma, mucinous carcinoma, Paget""s disease of the nipple, Phyllodes tumor, and tubular carcinoma can be detected. In addition, a breast cancer at any stage of progression can be detected, such as primary, metastatic, and recurrent breast cancer. Information regarding numerous types of breast cancer can be found, e.g., from the American Cancer Society (www3.cancer.org), or from, e.g., Wilson et al. (1991) Harrison""s Principles of Internal Medicine, 12th Edition, McGraw-Hill, Inc.